Due to their small size and relatively unsclerotized body, water
loss is a common problem facing all termite species, and they must
locate and utilize water resources to prevent or tolerate desiccation.
Water resources may be found as free water, moist food, or moist soil.
Termites live in various habitats and microhabitats with differing
levels of ambient humidity and availability of water, and water
management among termite species has been the subject of many studies
(Collins 1958, 1963, 1966, 1969; Abushama 1974; Rust et al. 1979;
Steward 1983; Sponsler & Appel 1990; Rudolph et al. 1990; Grube
& Rudolph 1999a, 1999b; Gallagher & Jones 2010; Gautam &
Henderson 2014). Some kalotermitid termites exhibit behavior and impacts
on survival that indicate that high humidity is toxic to them (Collins
1969; Minnick et al. 1973; Steward 1982, 1983; Rudolph et al. 1990; Wood
row et al. 2000). Strickland (1950), Collins (1958, 1969, 1991), and
Khan (1980), found that different termite species can tolerate various
(sometimes extreme) humidity and temperature conditions; however, there
are undoubtedly conditions that termites prefer over other conditions
that they can tolerate.

The first objective of this study was to investigate the
survivorship of 4 termite (Isoptera) species that inhabit distinctly
different microhabitats, including Coptotermes formosanus Shiraki
(Rhinotermitidae), Neotermes jouteli (Banks in Banks and Snyder)
(Kalotermitidae), Cryptotermes brevis Walker (Kalotermitidae), and
Cryptoterme cavifrons Banks (Kalotermitidae). Coptotermes formosanus and
N. jouteli live in high relative humidity (RH) environments, whereas Cr.
brevis is found in dry timber and Cr. cavifrons is found in natural wood
that can be wet or dry.

The second objective of this study was to investigate the RH
preferences of the aforementioned species. Humidity is not the only
water source available to termites. There are other water sources that
termites might use. These include metabolic water obtained through the
breakdown of food, from the fat body, free liquid water, as well as
water bound to various substrates (e.g., wood, soil, and nestmate
cadavers). Different termite species (i.e., drywood, dampwood,
subterranean) use or avoid certain environments with different moisture
and water sources. Whereas drywood termites depend heavily on
metabolically derived water, dampwood and subterranean termites that
live in high RH environments obtain water from their surroundings (Lee
& Wood 1971, Brammer & Scheffrahn 2007, Scheffrahn & Su
2007). The third objective of this study was to investigate whether
these 4 termite species use a variety of water sources for survival.

Materials and Methods

INSECT COLONIES

Individuals from colonies of C. formosanus, N.jouteli, Cr.
cavifrons, and Cr. brevis were collected in Broward County, Florida.
Different colonies of C.formosanus were collected from bucket traps as
described by Su and Scheffrahn (1986). Colonies of N. jouteli, Cr.
cavifrons, and Cr. brevis were collected from different pieces of wood.
Coptotermes formosanus and N. jouteli were kept in polystyrene boxes
(17.15 x 12.22 x 6.03 cm) with wood (Pinus sp.) maintained at 26.4
[degrees]C, and were regularly misted with water to maintain >95% RH.
Cryptotermes termites were kept in polystyrene boxes with wood pieces
and stored in the Incubator (26.4 [degrees]C and 41.5% RH). For Cr.
cavifrons, small dishes with water were provided. Populations of each
termite species survived well in these conditions and were used for
experiments as needed. Termites were kept in the incubator for <6 mo
before use.

TERMITE SURVIVORSHIP AT VARIOUS RELATIVE HUMIDITIES

Desiccative humidity chambers consisted of clear polystyrene boxes
(17.15 x 12.22 x 6.03 cm) with internal RH conditions stabilized using
three saturated salt solutions (i.e., NaCI, Mg[(N[O.sub.3]).sub.2], and
Mg[Cl.sub.2]), silica gel, and water (Fig. 1A) as described by Rockland
(1960), and Winston and Bates (1960). Open Petri dishes (93 mm diameter,
22 mm height) containing water or 1 of the saturated salt solutions were
placed at the center of 4 chambers (Fig. 1A, a-d), while silica gel was
placed on the bottom of the chamber (Fig. 1A, e) to produce the lowest
RH. The container edge was coated with petroleum jelly (Eboneen Products
Company, Inc., Hartford, Connecticut) to create a seal between the lid
and chamber. Temperature and humidity levels were measured daily using a
probe (THW3; Amprobe, Everett, Washington) (Fig. 1B, i) through an
access hole (1.59 cm diameter) in the lid. These holes were plugged with
rubber stoppers while not in use (Fig. 1B, h). The average RH ([+ or -]
standard error of the mean [SEM]) of the chambers and their associated
stabilizing material were as follows (n = 73): 92.0 [+ or -] 0.07%
([H.sub.2]O), 72.9 [+ or -] 0.08% (NaCI), 55.7 [+ or -] 0.09%
(Mg[(N[O.sub.3]).sub.2]), 34.3 [+ or -] 0.04% (Mg[Cl.sub.2]), and 18.2
[+ or -] 0.14% (silica gel). The average temperature ([+ or -]SEM) of
the chambers and their associated stabilizing material were as follows
(n = 73): 23.5 [+ or -] 0.06 [degrees]C ([H.sub.2]O), 23.6 [+ or -] 0.06
[degrees]C (NaCl), 23.5 [+ or -] 0.06 [degrees]C
(Mg[(N[O.sub.3]).sub.2]), 23.7 [+ or -] 0.06 [degrees]C (MgCLJ, and 23.6
[+ or -] 0.06 [degrees]C (silica gel). Groups of 10 termite individuals
per species were placed in the Petri dish (35 mm diameter, 10 mm height)
of each chamber with small holes cut into the lids of the dishes to
allow air movement and yet prevent termite escape. Dishes were also
provisioned with a small (15 x 15 x 9 mm) piece of wood (Pinus sp.) as a
food source. Four dishes, each containing 1 of the 4 termite species,
were placed in the humidity chamber surrounding water or salt solutions
(Fig. 1A, a-d), or on top of the silica gel in a chamber (Fig. 1A, e).

Temperature and humidity readings were recorded every 24 h
post-introduction for 12 d. Daily counts of live termites were recorded
for a 12 d period. A follow-up experiment also was conducted to examine
recovery of N. jouteli individuals that exhibited visible reduction in
body mass. At the conclusion of the 12 d study, the surviving N. jouteli
Individuals from the various RH were weighed and then placed in a Petri
dish and provided with food and water for 1 wk. Surviving termites were
counted and weighed again to examine recovery when provided with
adequate food and water resources.

Statistical analysis was carried out using JMP statistical software
(SAS 2015). An analysis of variance (ANOVA) for a 4 by 5 factorial
experimental design was conducted to test for differences. Termite
species (4) and RH (5) were the factors, and percentage survival was the
response variable. Percentage survival values were arcsine-square root
transformed before analysis. Post hoc Tukey honest significant
difference tests were used to evaluate all pairwise differences at a =
0.05. Data from 6 replicates were analyzed.

Chambers housed modified plastic vials (25 mm inner diameter, 15 mm
height) cut to hold the termites while preventing their escape (Fig. 2C,
k). A hole (6 mm diameter) was drilled into the wall of each of the 4
radial chambers (2.5 cm from base) to allow pieces of plastic tubing
(5.5 mm diameter, 50.0 mm length) (Fig. 2C, j) to connect radial termite
vials to the vial in the central chamber. All termite vials were
suspended by plastic tubes above the stabilizing materials. Groups of 25
pseudergates were placed into the introduction chamber for the larger
sized species N. jouteli, while 50 termites (pseudergates and workers)
were used for the other 3 smaller sized species.

Termites could move and acclimate to any chamber for 12 to 16 h.
Black satin cloth covered the arenas to provide darkness. Counts of
termites within each chamber were then recorded. Termites found in the
connecting tubes were included in the counts for the connected chamber.
Statistical analysis was conducted using JMP statistical software (SAS
2015). A 4 by 5 factorial analysis was conducted with termite species
(4) and RH (5) as the factors and percentage of termites found in a
chamber as the response variable. The percentage values were
arcsine-square root transformed before the analysis of variance (ANOVA)
was used to test whether there was a significant difference among RH
preference for the 4 species. Post hoc Tukey honest significant
difference tests were used to separate the differences for all pairs at
a = 0.05. Data from 6 replications were analyzed.

TERMITE SURVIVORSHIP WITH VARIOUS TYPES OF WATER SOURCES

Experimental chambers were constructed using clear plastic jars (52
mm diameter, 53 mm height) with plastic lids and lid liners. The bottoms
of these jars were scratched with sandpaper to provide traction for
termite movement. Temperature and humidity levels inside the jars were
measured as described previously.

Five chambers that provided various sources of water, or the lack
thereof, were used for the experiment. These included: chambers with a
piece of dry wood (Pinus sp.; 18 x 18 x 8 mm), a piece of dry wood with
a humidity source (i.e., a 2 x 6 cm wetted filter paper strips attached
to the upper inner side of the chamber), a piece of wet wood, a piece of
dry wood with wet sandy soil (5 g sand + 1.5 mL deionized water), and a
piece of dry wood with a source of free water in a small dish (modified
shell vial cap, 15 mm diameter, 3 mm height). Water was added as needed
to maintain RH and free water levels. The average (n = 48) RH ([+ or -]
SEM) of the chambers and their associated water sources were as follows:
91.0 [+ or -] 0.22% (wet wood), 49.6 [+ or -] 0.61% (dry wood), 92.6 [+
or -] 0.22% (wet soil), 91.1 [+ or -] 0.22% (wood + water), and 94.2 [+
or -] 0.18% (wood + RH). The average (n = 48) temperature ([+ or -] SEM)
of the chambers and their associated water sources were as follows: 24.2
[+ or -] 0.08 [degrees]C (wet wood), 24.1 [+ or -] 0.08 [degrees]C (dry
wood), 24.2 [+ or -] 0.07 [degrees]C (wet soil), 24.2 [+ or -] 0.07
[degrees]C (wood + water), and 23.0 [+ or -] 0.03 [degrees]C (wood +
RH).

Three sets of each of the chambers were broken down weekly for 4 wk
to record termite survival for each species using destructive sampling.
The data were analyzed using JMP statistical software (SAS 2015) and a 4
by 5 factorial experimental design with termite species (4) and water
source (5) as the factors, and percentage survival as the response
variable. Percentage survival data were arcsine square root transformed
before analysis. Weekly data were analyzed separately. ANOVA was
conducted to test for significant differences and post hoc Tukey honest
significant difference tests were used to separate the differences for
all pairs at a = 0.05. Data from 3 replications were analyzed.

Results

TERMITE SURVIVORSHIP AT VARIOUS RELATIVE HUMIDITIES

Termites displayed statistically different responses to RH (F =
90.0; df = 3, 116; P <0.001). There was no significant difference in
survival at any RH (18.2-92.0%) for Cr. cavifrons, Cr. brevis, and N.
jouteli, but there was a significant difference for C. formosanus. This
species survived at 92.0% RH, but died within a few days when maintained
at <72.9% RH (Table 1). However, N. jouteli individuals in all the
humidity chambers (18.2-72.9% RH) except the water chamber (92.0% RH)
were visibly smaller at the end of the experiment than at the start. The
Cryptotermes species did not exhibit this change in size. At 18.2 to
72.9% RH levels, N. jouteli groups decreased in mass (4.4-151.2 mg loss
per 10 termites). Of the 180 N. jouteli individuals that survived at the
various RH and were then placed in a dish and provided with food and
water, 172 recovered. These 172 individuals exhibited a total weight
gain of approximately 327 mg. The remaining 8 individuals were not
accounted for, indicating they may have been cannibalized by nestmates
as a food and water source. The N. jouteli groups of 10 individuals
exhibited a decrease in mass of approximately 42.0% of their weight,
either due to the loss of body water or body fat.

TERMITE PREFERENCE FOR VARIOUS RELATIVE HUMIDITIES

Termites displayed statistically different responses to RH (F =
7.06; df =3,116; P<0.001). The wetwood (nests in wood or soil high in
moisture) species of C formosanus and N. jouteli preferred the highest
RH of 90.7% RH, which was significantly more than Cr. cavifrons and Cr.
brevis and significantly more than the other RH (Table 2). The
Cryptotermes species did not exhibit a preference for any RH when
compared among species and among RH.

TERMITE SURVIVORSHIP WITH VARIOUS TYPES OF WATER SOURCES

Termites displayed statistically different responses to RH for wk 1
(F = 5.71; df = 3, 56; P <0.001), wk 2 (F = 3.30; df = 3, 56; P
<0.001), wk 3 (F = 3.89; df = 3, 56; P <0.001), and wk 4 (F =
4.91; df = 3, 56; P <0.001). Within a week, none of the C. formosanus
individuals survived in the chambers provisioned with a piece of dry
wood at RH <49.6%. Survival of C. formosanus was significantly lower
in the dry wood chamber when compared to the other 3 species at all
weeks. They survived significantly better throughout the 4 wk experiment
when provided with some form of water at RH >90% (Table 3) when
compared to the low humidity of the dry wood chamber. The only exception
was the low survivorship of 33.3% when C. formosanus was provided with a
piece of dry wood and free water at 91.1% RH for 4 wk. Because of the
high survivorship of 96.7% at 4 wk when provided with dry wood at a
slightly elevated RH (94.2%), the low 33.3% survivorship may be
attributed to other factors such as handling. In the first 3 weeks,
survival was not significantly different for N. jouteli, Cr. Brevis, and
Cr. cavifrons when compared between the 5 water source chambers, as well
as when compared with each other. After 4 wk, survival of C. formosanus
exposed to dry wood was significantly lower when compared to wet wood,
wet soil, and wood with a RH source. Cryptotermes brevis also exhibited
significantly lower survival with wet wood when compared to C.
formosanus, and wood with a RH source when compared to Cr. cavifrons
(Table 3).

Discussion

Results of this study showed that C formosanus is less capable of
tolerating desiccation than N. jouteli, Cr. cavifrons, or Cr. brevis.
This agrees with previous studies of Collins (1958, 1963, 1966, 1969)
and Khan (1980) on Kalotermitidae and Rhinotermitidae species. Neotermes
jouteli did not express a difference in survival at the various RH, but
lost body mass while remaining alive, most likely due to the need to
generate metabolic water from reserve body fat, and exhibited a
reduction in movement at RH below 72.9%.

The Cryptotermes species did not show a difference in survival when
compared at the 5 RH of the first objective and did not exhibit a change
in behavior. The physiological mechanisms involved in desiccation
tolerance remain to be elucidated, but are probably related to cuticular
permeability and percentage of total body water.

The results illustrate the necessity for C. formosanus, and
preference of N. jouteli, to inhabit environments with high levels of
moisture. Coptotermes formosanus, a subterranean species, not only
requires high RH, but also other sources of water for normal activity
and survival (Gautam & Henderson 2011). Tunneling activity of
Coptotermes gestroi (Wasmann) (Rhinotermitidae), Heterotermes tenuis
(Hagen) (Rhinotermitidae), C formosanus, and Reticulitermes flavipes
(Kollar) (Rhinotermitidae) in studies by Arab and Costa-Leonardo (2005)
and Su and Puche (2003), showed that these species tunneled more in
substrates with higher moisture content. Additionally, the effect of
substrate and food moisture levels on survival, consumption, and
distribution of Microcerotermes crassus Snyder (Termitidae), Macrotermes
carbonarius (Hagen) (Termitidae), Macrotermes gilvus (Hagen)
(Termitidae), C. gestroi, C. formosanus, Reticulitermes speratus (Kolbe)
(Rhinotermitidae), R. flavipes, Reticulitermes tibialis Banks in Banks
and Snyder (Rhinotermitidae), and Reticulitermes virginicus (Banks)
(Rhinotermitidae) also highlights the importance of adequate water
availability for other species (Delaplane & La Fage 1989; Nakayama
et al. 2004, 2005; Green et al. 2005; McManamy et al. 2008; Wong &
Lee 2010; Hu et al. 2012). Many termites (including C. formosanus and N.
jouteli) are more likely to be found, survive longer, and consume food
resources at higher rates in environments in which water resources are
more likely to persist, but some, such as Cr. brevis, are not.

As reported for Kalotermitidae and Termopsidae by Collins (1991),
the dampwood termite, N. jouteli, lives within its food source and
requires moist conditions. However, N. jouteli and Cr. cavifrons inhabit
wood pieces that are more likely to be subject to RH changes. Given that
their geographic ranges include areas with extended wet and dry seasons,
it is beneficial for them to be able to tolerate a range of RH and water
availabilities. In this study, Cr. cavifrons and N. jouteli were able to
tolerate conditions in which water sources were lacking, as well as
utilize water sources when they were present.

As was noted by Pence (1956) and Collins (1958), the Cryptotermes
species in this study were also less active than the subterranean and
dampwood species, and tended to aggregate with little movement unless
disturbed. Coptotermes formosanus and N. jouteli, while more active,
also tended to aggregate. This aggregative huddling was likely a
behavioral mechanism to prevent desiccation from water loss through
evaporation from the body by decreasing overall surface area of the
group. This behavior, termed the "group effect" by Grasse and
Chauvin (1944), was reported to increase termite survival for several
species (Pence 1956; Sen-Sarma & Chatterjee 1966; Minnick et al.
1973; Abushama 1974; Ahmad et al. 1982; Malik & Sheikh 1990; Cabrera
& Rust 1996).

Another group behavior involved in desiccation tolerance is the
consumption of nestmates. Cannibalism, as described by Collins (1991),
was not explicitly observed in this study, but the fact that there were
individuals missing from the Neotermes recovery study suggests they were
probably cannibalized. This behavior is an additional means of obtaining
water and food resources under water-stressed conditions.

Whereas some termites require access to water sources, too much
moisture can be detrimental to other species. While each week of
exposure was not statistically compared to the other weeks, there was a
decrease in average survival from week 1 to 4 for Cr. brevis. There was
also a decrease in survival for Cr. brevis when exposed to environments
with RH >90%, and indicated that avoiding prolonged contact with free
water is important to survival in this species. Our results agree with
previous works of Collins (1969), Minnick et al. (1973), Steward (1982,
1983), Rudolph et al. (1990), and Woodrow et al. (2000), in which they
found evidence of water toxicity in drywood termite species. If
individuals of cr. brevis could avoid direct contact with a water
source, such as on the wood block provided, they were generally better
able to tolerate these less-than-favorable environments with extended
exposure.

Results of this study highlight the importance of water to the
survival of four termite species and the differences in their RH
preference, desiccation and RH tolerance, and moisture exposure
tolerance. Further studies are needed to examine their morphological and
physiological characteristics such as cuticular structure, spiracular
openings, and cuticular permeability that may contribute to these
differences.

Acknowledgments

We are grateful to A. Mullins (University of Florida) for review of
this article and R. Pepin for technical support. This manuscript is a
portion of a dissertation submitted by J. Zukowski in partial
fulfillment of the requirements for a Ph.D. at the University of
Florida. This work was supported in part by the USDA National Institute
of Food and Agriculture, Hatch project number FLA-FTL-005342. Additional
funding was provided by USDA-ARS under the grant agreement No.
58-6435-8-108.

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